Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: a comparison

Two different forms of acid invertase (EC 3.2.1.26) were extracted from expanding leaves of the stinging nettle (Urtica dioica L.). One form was soluble and could be localized within the cytosol, whereas the other was ionically bound to the cell-wall and could not be detected in protoplasts. Both forms were purified, the latter to homogeneity. Western blotting with antibodies against the pure enzyme from cell walls was positive with the cell-wall enzyme but negative with the soluble form of acid invertase. Both forms are glycoproteins with identical molecular weights of 58 kDa. The K_m values for sucrose (raffinose) are 5 mM (4.8 mM) for the soluble and 1.2 mM (3.6 mM) for the cell-wall-bound enzyme. The pH optimum of the latter is slightly more acidic (4.5) than that of the soluble invertase (5.5). Both forms could easily be distinguished by their isoelectric points which were determined at pH 4.6 for the soluble and pH 9.3 for the wall-bound enzyme. When extraction and purification were carried out in the absence of protease inhibitors, both acid invertases showed microheterogeneity (multiple forms). However, with benzamidine and phenylmethylsulfonylfluoride as protease inhibitors each invertase produced only one protein band upon isoelectric focusing and gel electrophoresis, respectively.Abbreviations B benzamidine - Con A concanavalin A - FPLC fast protein liquid chromatography - IEF isoelectric focusing - kDa kilodalton - pI isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the Sonderforschungsbereich 137.     

Production,IMAC purification,and molecular modeling of N-carbamoyl-D-amino acid amidohydrolase C-terminally fused with a six-his peptide

A six-His peptide was genetically engineered to the C-terminus of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase monomer to facilitate the protein purification with immobilized metal affinity chromatography (IMAC). The fusion enzyme, named as DCaseH, was overexpressed in Escherichia coli and one-step IMAC-purified. The production study showed that DCaseH was optimally produced at 15 degrees C for 25 h by the induction of 0.05 mM IPTG. Both Co(2+)-chelated TANOL gels and Ni(2+)-chelated nitriloacetic acid agarose gels efficiently purified DCaseH, with the former yielding purer enzyme than the latter. Highly pure DCaseH was obtained in the former purification with the addition of 5 mM imidazole in the washing buffer, and the specific enzyme activity was increased more than 11-fold. Denaturing IMAC purification successfully purified DCaseH from inclusion bodies that were mostly composed of the overexpressed DCaseH, while the attempt to refold the purified enzyme by either dialysis or solid-state refolding was not achieved. The purified native enzyme was optimally active at pH 6.5 and 50 degrees C, and the presence of 10% glycerol increased the activity. The molecular modeling of dimeric DCaseH indicated that the six-His tags were freely exposed to the protein surface, resulting in the selective and effective IMAC purification of DCaseH.     

An extracellular polysaccharide produced by Zoogloea ramigera 115

A weakly acidic polysaccharide was purified from the extracellular zoogloeal matrix produced by Zoogloeal ramigera 115. The purified polysaccharide was homogeneous as judged by sedimentation analysis, and the average molecular weight was estimated to be about 10(5) by gel permeation chromatography of the fully methylated preparation. The polysaccharide was composed of D-glucose, D-galactose and pyruvic acid in an approximate molar ratio 11:3:1.5. On the basis of methylation, periodate oxidation, Smith degradation and partial hydrolysis, the following highly branched structure was deduced for the polysaccharide: a long chain mainly consisting of beta 1 leads to 4-linked glucose residues branching at the C-3 or C-6 position of galactose residues which are present in beta 1 leads to 4 or beta 1 leads to 3 linkages as the minor component of the long chain; pyruvic acid residues, the sole acidic component, are linked to the nonreducing end and/or 1,3-linked glucose residues through 4,6-ketal linkages. The purified polysaccharide was not readily soluble in water and had a high affinity for several metallic ions (e.g, 0.25 mumol Fe3+/mg, and 0.17 mumol Fe2+ mg). Upon addition of metallic ions (1 mM) to a gelatinous aqueous solution of the polysaccharide (K+ form, 0.125%), more than 80% of it immediately coprecipitated out with them.     

Isolation of a coaggregation-inhibiting cell wall polysaccharide from Streptococcus sanguis H1.

Coaggregation between Streptococcus sanguis H1 and Capnocytophaga ochracea ATCC 33596 cells is mediated by a carbohydrate receptor on the former and an adhesin on the latter. Two methods were used to release the carbohydrate receptor from the gram-positive streptococcus, autoclaving and mutanolysin treatment. The polysaccharide released from the streptococcal cell wall by either treatment was purified by ion-exchange chromatography; this polysaccharide inhibited coaggregation when preincubated with the gram-negative capnocytophaga partner. After hydrolysis of the polysaccharide by hydrofluoric acid (HF), the major oligosaccharide of the polysaccharide was purified by high-performance liquid chromatography. By analysis of the HF hydrolysis of the polysaccharide and the purified oligosaccharide, this major oligosaccharide appeared to be the repeating unit of the polysaccharide, with minor components resulting from internal hydrolysis of the major oligosaccharide. Gas chromatography results showed that the oligomer was a hexasaccharide, consisting of rhamnose, galactose, and glucose, in the ratio of 2:3:1, respectively. By weight, the purified hexasaccharide was a fourfold-more-potent inhibitor of coaggregation than the native polysaccharide. Resistance to hydrolysis by sulfuric acid alone and susceptibility to hydrolysis by HF suggested that oligosaccharide chains of the polysaccharide are linked by phosphodiester bonds. Studies with a coaggregation-defective mutant of S. sanguis H1 revealed that the cell walls of the mutant contained neither the polysaccharide nor the hexasaccharide repeating unit. The purification of both a polysaccharide and its constituent hexasaccharide repeating unit, which both inhibited coaggregation, and the absence of this polysaccharide or hexasaccharide on a coaggregation-defective mutant strongly suggest that the hexasaccharide derived from the polysaccharide functions as the receptor for the adhesin from C. ochracea ATCC 33596.     

The Metabolism of Proline in Higher Plants: II. L-PROLINE DEHYDROGENASE FROM COTYLEDONS OF GERMINATING PEANUT (ARACHIS HYPOGAEA L.) SEEDLINGS

The L-proline-dependent reduction of NAD⁺ has been obtainedwith a soluble enzyme extracted from acetone powders of thecotyledons of 3- to 5-day-old germinating peanut seedlings.The enzyme has been purified approximately 20-fold. NAD⁺ ismuch more effective as an electron acceptor than NADP⁺, thereaction rate with the latter being only 15 per cent that withthe former. The K_m for L-proline at pH 10.3, with NAD⁺ saturating,is 0.30 mM, and that for NAD⁺, with L-proline saturating, is0.25 mM. NADP⁺ is an excellent competitive inhibitor for NAD⁺with a K₁ of 6.2 µM. L-proline, L-proline methyl ester, and 3,4-dehydro-DL-prolineare equally effective as substrates. Thiazolidine-4-carboxylatecatalyses the reduction of NAD⁺ at 63 per cent the rate withL-proline. D-proline is not a substrate nor an inhibitor. L-prolineamide has 11 per cent the activity of L-proline and N-methyl-L-prolinehas a very slight activity. Other proline derivatives or thelower and higher homologues are completely inactive. Incubation with L-proline-¹⁴C in the presence of NAD⁺ yieldsone product which has a higher Rf than proline using butanol-aceticacid-water as the solvent in paper chromatography. Elution ofthis product and treatment with hydrogen peroxide gives severalproducts of high Rf with the same solvent mixture. None of theproducts is -aminobutyrate or glutamic acid. This eliminateseither P2C or P5C as the reaction product.     

Two types of rat gastric mucus glycoprotein subunits

Gastric mucus glycoproteins were extracted with 2% Triton X-100 from rat gastric corpus and antrum and purified by CsCl equilibrium centrifugation. Corpus mucus glycoproteins were degraded into what appeared to be two "subunits" (Mw 4.4 x 10(5) and 6 x 10(6)) by the reduction of disulfide bonds. Papain digestion of the latter produced glycopeptides with a molecular weight of approximately 4.4 x 10(5). This type of subunit had carbohydrate chains with about 9 sugars attached to every 2 amino acid residues. Papain digestion of the former type of subunit revealed no change in the elution profile on Bio-Gel A-15m. This type of subunit had carbohydrate chains with 17-19 sugars attached to every 3 amino acid residues. The subunit of antral mucus glycoproteins was essentially the same as the former type of corpus subunits in molecular weight (Mw 4.4 x 10(5)) and average oligosaccharide chain length. These results suggest that there are two distinct types of mucus glycoprotein subunits in rat stomach.     

Isolation and characterization of a succinylated polysaccharide from the cell wall of Micrococcus agilis

A polysaccharide consisting of rhamnose, galactose, glucosamine and ester-linked succinic acid was extracted from the isolated cell walls of Micrococcus agilis by the hot water-phenol and 5% trichloroacetic acid (TCA) extraction methods. The hot water-phenol extractable polysaccharide accounted for 30% of the weight of the wall, with 23% by the TCA method. Phosphorus contents were less than 0.01% of the polysaccharide. Succinyl residues released by alkali treatment (0.1 N NaOH, 30 min, 37 degrees C) were identified by gas-liquid chromatography, and accounted for 6.3% and 5.1% of the polysaccharide purified from the hot water-phenol and TCA extracts, respectively. The polysaccharide was not bound when chromatography on Concanavalin A-Sepharose 4B (Con A/Sepharose 4B) columns was performed and it could thus be separated from any residual membrane lipomannan. The purified polysaccharide behaved as a negatively-charged polymer on electrophoresis in 1% agarose (at pH 8.6). A strong cross-reaction, unaffected by removal of the succinyl groups, was observed with type XXIII pneumococcal polysaccharide antiserum indicating the presence of L-rhamnose, linked through non-reducing, lateral end groups.     

Purification and Properties of Extracellular Amylase from the Hyperthermophilic Archaeon Thermococcus profundus DT5432

A hyperthermophilic archaeon, Thermococcus profundus DT5432, produced extracellular thermostable amylases. One of the amylases (amylase S) was purified to homogeneity by ammonium sulfate precipitation, DEAE-Toyopearl chromatography, and gel filtration on Superdex 200HR. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 5.5 to 6.0 and was stable in the range of pH 5.9 to 9.8. The optimum temperature for the activity was 80(deg)C. Half-life of the enzyme was 3 h at 80(deg)C and 15 min at 90(deg)C. Thermostability of the enzyme was enhanced in the presence of 5 mM Ca(sup2+) or 0.5% soluble starch at temperatures above 80(deg)C. The enzyme activity was inhibited in the presence of 5 mM iodoacetic acid or 1 mM N-bromosuccinimide, suggesting that cysteine and tryptophan residues play an important role in the catalytic action. The amylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen to produce maltose and maltotriose of (alpha)-configuration as the main products. Smaller amounts of larger maltooligosaccharides were also produced with a trace amount of glucose. Pullulan; (alpha)-, (beta)-, and (gamma)-cyclodextrins; maltose; and maltotriose were not hydrolyzed.     

Fruit Softening II. Precursor Incorporation into Pectin by Pear Tissue 6

The incorporation of radioactivity from precursors of methylester and galacturonosyl residues into pectin was investigatedusing tissue slices cut from ripening pear fruits. Incorporationfrom ¹⁴CH₃ methionine into methyl ester of water soluble pectinincreased 10 fold in 4 d at 18 °C and declined in laterstages of ripening. Activity from [³H]inositol could not bedetected in gaJacturonic acid released enzymically from solublepolysaccharides. When l³H]glucose was used as a precursor, activitycould be detected in galacturonic acid released from both thesoluble and insoluble polysaccharide fractions. Methionine wasa more efficient precursor of methyl ester groups than S-adenosylmethionine or S-methyl methionine; incorporation from all threeprecursors was inhibited under nitrogen. Radioactively labelledmethyl ester did not decline during a 225 min ‘chase’following a 15 min ‘pulse’ of [¹⁴CH₃]methionine;the total pectin content of slices increased by 20% during this4 h incubation.     

Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes

The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75 mumol/min/mg). Palmitoyl-DHAP competitively inhibited the reduction of hexadecyl-DHAP, indicating that the same enzyme catalyzes the reduction of both acyl-DHAP and alkyl-DHAP. NADH can substitute for NADPH, but the Km of the enzyme for NADH (1.7 mM) is much higher than that for NADPH (20 microM). The purified enzyme is competitively (against NADPH) inhibited by NADP+ and palmitoyl-CoA. The enzyme is stable on storage at 4 degrees C in the presence of NADPH and dithiothreitol.     

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.     

Immobilization and properties of Leuconostoc mesenteroids dextransucrase

Dextransucrase from Leuconostoc mesenteroides (NRRL B-512F) was purified by ultrafiltration and gel filtration chromatography in 54% yield. The specific activity of a heart cut was 58.6 U/mg; cumulative purification of that preparation was 247?fold. Of 13 carriers surveyed, only alkylamine porous silica gave immobilization efficiencies consistently above 15 %. Immobilization to silica changed the properties of dextransucrase relatively little, the optimum pH for activity remaining at 5.2, while that for stability decreased from pH 5.5?6 to pH 5.2. In short assays, highest activities of both soluble and immobilized dextransucrase occurred at 30°C. Activation energies below that temperature were 8.6 kcal/mol for the former form and 1.7 kcal/mol for the latter. Maximum stabilization of soluble dextransucrase was attained by 5mM Ca²⁺.     

Rabbit renal cortical microsomes metabolize arachidonic acid to trihydroxyeicosatrienoic acids

(1-¹⁴C) Eicosatetraenoic (Arachidonic) acid was incubated wiht microsomes from rabbit renal cortex and NADPH (1 mM) for 15 min at 37°C. The products were extracted and purified by high pressure liquid chromatography. Some of the most polar metabolites were identified by gas chromatography mass spectrometry. They were 11, 12, 19- and 11, 12,20-trihydroxy-5,8-14-eicosatrienoic acid, 14,15,19- and 14,15,20- trihydroxy-5,8,11-eicosatrienoic acid, and 11,12-dihydroxy-19-oxo- 5,8,14-eicosatrienoic acid. These products were likely formed by ω- and (ω−1)-hydroxylation of 11,12-dihydroxy-5,8,14-eicosatrienoic aic and 14,15-dihydroxy-5,8,11-eicosatrienoic acid, two recently identified metabolites of arachidonic acid in fortified rabbit kidney microsomes.     

Purification, Kinetic and Regulatory Properties of Phosphofructokinase from Chiorella pyrenoidosa

Phosphofructokinase was purified 585-fold from Chlorella pyrenoidosaby using a combination of ammonium sulphate fractionation, filtrationthrough Sepharose 4B and chromatography on DEAE-Sephacel. Enzymestability was maintained by the presence of 50 mM P_i at pH 6.6.The optimum pH for activity was 7.7. Concentrations of substratesrequired to achieve half maximal velocity in the standard assaywere 9 µM (ATP) and 0.2 mM (fructose-6-P). ATP above 0.5mM was inhibitory. Enzyme activity was inhibited by high concentrations(10–100 mM) of P_i but lower concentrations (1–5mM) were effective in relieving the influence of other inhibitorssuch as P enolpyruvate. Inhibition by P-enolpyruvate was greaterat lower pH and with less P_i in reaction mixtures: 50% inhibitioncould be attained with 0.1 mM P-enolpyruvate. Fructose-2,6-bisphosphate,which was shown to be present in Chlorella, had no effect onthe phosphofructokinase. Chlorella appeared to contain onlyone form of phosphofructokinase, possibly in the chloroplast.No pyrophosphate :D-fructose-6-P 1-phospho transferase activitycould be detected. (Received February 20, 1984; Accepted December 5, 1984)     

A wounding-induced PPO from cowpea (Vigna unguiculata) seedlings

Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and congruent with220kDa) were detected by partially denaturing SDS-PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH(4))(2)SO(4) precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors - resorcinol, EDTA, sodium azide and tropolone - of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol (K(m)=9.86mM, V(max)=24.66 EU [DeltaAmin(-1)]) and catechol (K(m)=3.44mM, V(max)=6.64 EU [DeltaAmin(-1)]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 degrees C. The most effective inhibitor was tropolone, whereas addition of 10mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.     

Isozymes of Superoxide Dismutase in Chlamydomonas and Purification of One of the Major Isozymes Containing Fe

Electrophoretic analysis of Chlamydomonas reinhardtii extractrevealed at least 4 distinct superoxide dismutase (SOD) activitybands as well as several additional minor bands. Among them,one was deduced to be Fe-type and the other three Mn-type basedon their susceptibility to KCN and H₂O₂. The Fe-SOD, which occupiedabout 40% of the total soluble activity, was purified to homogeneityusing ammonium sulfate fractionation followed by DEAE-cellulose,hydroxyapatite, and Superdex 75 gel-permeation chromatography.The 40-kDa native enzyme was composed of two identical 20-kDasubunits with a low shoulder of absorption at {small tilde}350nm. The NH₂-terminal amino acid sequence determined up to residue29 showed a high homology to those of Fe-SOD from Arabidopsisthaliana, Glycine max, and Nicotiana plumbaginifolia. (Received December 21, 1992; Accepted May 28, 1993)     

Characterization of cytosolic sialidase from Chinese hamster ovary cells: part I: cloning and expression of soluble sialidase in Escherichia coli

The cDNA of Chinese hamster ovary (CHO) cell cytosolic sialidase was amplified by RT-PCR and cloned into the pGEX-2T plasmid vector encoding for glutathione S-transferase (GST). Screening revealed transformed Escherichia coli clones with the constructed plasmid encoding the CHO cell sialidase sequence. After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, SDS-PAGE of the total protein extracts revealed a new protein of about 70 kDa, correlating with the molecular weight of a fusion protein composed of the GST (26 kDa) and the cloned cytosolic CHO cell sialidase (43 kDa). A soluble fusion protein was purified from sonified E. coli homogenates by one-step affinity chromatography on Glutathione Sepharose 4B, which showed sialidase activity towards 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (MUF-Neu5Ac) substrate. Induction of cells with 0.1, 0.5, and 1.0 mM IPTG revealed highest total protein amounts after induction with 1.0 mM IPTG, but highest specific activity for affinity chromatography purified eluates from cultures induced with 0.1 mM IPTG. Therefore, large scale production was performed by inducing cells during exponential growth in a 25 L bioreactor for 3 h with 0.1 mM IPTG after chilling the cell suspension to 25 degrees C. The amount of 26.46 mg of 40-fold purified GST-sialidase with a specific activity of 0.999 U/mg protein was obtained from crude protein extracts by one-step affinity chromatography. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) and Neu5Ac were competitive inhibitors for the sialidase, the former being the more effective one using MUF-Neu5Ac as the substrate. The cytosolic sialidase is capable of desialylating a wide spectrum of different types of gangliosides using a thin-layer chromatography overlay kinetic assay without detergents. This is the subject of the accompanying paper (Müthing, J.; Burg, M. Carbohydr. Res. 2001, 330, 347-356).     

A rat liver lysosomal membrane flavin-adenine dinucleotide phosphohydrolase: purification and characterization

An enzyme hydrolyzing flavin-adenine dinucleotide (FAD) to flavin mononucleotide and AMP was identified and purified from rat liver lysosomal (Tritosomal) membranes. The purified enzyme showed a single band on silver-stained denaturing gels with an apparent Mr 70,000. Periodate-Schiff staining after denaturing gel electrophoresis of whole membrane preparations revealed that this enzyme is one of the major glycoproteins in lysosomal membranes. FAD appeared to be the preferred substrate for the purified enzyme; equivalent concentrations of NAD or CoA were hydrolyzed at about one-half of the FAD rate. Negligible activity (less than or equal to 16%) was noted with ATP, TTP, ADP, AMP, FMN, pyrophosphate, or p-nitrophenylphosphate. The enzyme was inhibited by EDTA or dithiothreitol. It was stimulated by Zn, and was not affected by Ca or Mg ions, nor by p-chloromercuribenzoate. The pH optimum for FAD hydrolysis was 8.5-9 with an apparent Km of 0.125 mM. Antibodies prepared against the purified enzyme partially (50%) inhibited FAD phosphohydrolase activity in lysosomal membrane preparations but had no effect on the soluble lysosomal acid pyrophosphatase known to hydrolyze FAD. This enzyme could not be detected immunochemically in preparations of microsomes, Golgi, plasma membranes, mitochondrial membranes, or the soluble lysosomal fraction, suggesting that the enzyme is different from either soluble lysosomal acid pyrophosphatase or other FAD hydrolyzing activities in the liver cell.     

Nitric oxide enhances hydrogen peroxide-mediated endothelial permeability in vitro

The objective ofthis study was to evaluate the effects of nitric oxide (NO) onH₂O₂-mediatedendothelial permeability.H₂O₂ (0.1 mM) increased permeability at 90 min to 298% of baseline. Spermine NONOate (SNO), an NO donor, at 0.1 or 1 mM did not alter permeability. However, 0.1 mMH₂O₂ + 1 mM SNO increased permeability to 764%, twice that of 0.1 mMH₂O₂alone. These treatments were not directly toxic to endothelial cells.This NO effect was concentration dependent, inasmuch as 0.1 mM SNO didnot significantly change H₂O₂-mediatedpermeability. The NO-enhanced,H₂O₂-dependentpermeability required the simultaneous presence of NO andH₂O₂,inasmuch as preincubation with SNO for 30 min followed by 0.1 mMH₂O₂did not alter permeability. Staining of endothelial junctions showed widening of the intercellular space only in junctions of cells exposedtoH₂O₂(0.1 mM) + SNO (1 mM). Furthermore, NO did not affectH₂O₂metabolism by endothelial cells but significantly depletedintracellular glutathione. This reduction of cell glutathione producedby NO exposure recovered 15-30 min after removal of the NO donor.NO-enhanced permeability was completely blocked by methionine (1 mM), ascavenger of reactive oxygen species, and by the iron chelatordesferrioxamine (0.1 mM). These results suggest that NO may exacerbatethe effects ofH₂O₂-dependentincrease in endothelial monolayer permeability via the iron-catalyzedformation of reactive oxygen metabolites.

     

ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells

Inwardlyrectifying K⁺ current(I_Kir) infreshly isolated bovine retinal pigment epithelial (RPE) cells wasstudied in the whole cell recording configuration of the patch-clamptechnique. When cells were dialyzed with pipette solution containing noATP, I_Kir randown completely in <10 min [half time(t_1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustainedI_Kir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mMATP was used,I_Kir ran down by~71%. Mg²⁺ was a criticalcofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg²⁺(t_1/2 = 1.8 min).I_Kir also randown when the pipette solution contained 4 mMMg²⁺ + 4 mM5'-adenylylimidodiphosphate(t_1/2 = 2.7 min)or 4 mM adenosine 5'-O-(3-thiotriphosphate)(t_1/2 = 1.9 min),nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. Weconclude that the sustained activity ofI_Kirin bovine RPE requires intracellular MgATP and that the underlyingmechanism may involve ATP hydrolysis.